Many pathogenic bacteria use a specialized secretion system to inject virulence proteins directly into the cells they infect. The injected proteins, by mimicking host-cell mechanisms, can then subvert normal cellular function. The type III secretion system (TTSS) is a sophisticated protein complex with an overall shape similar to a hypodermic needle. More than twenty unique types of proteins are required for its assembly, most of which are found among a wide variety of animal as well as plant pathogens. Electron microscopy has sketched the broad outlines of TTSS structure, but it does not have sufficient resolution to reveal the details required to understand, and eventually inhibit, the needle's function. At the ALS, researchers from Canada and the U.S. performed crystallographic studies of EscJ, the protein that makes up the needle's ring-shaped base. Their analysis of the EscJ ring not only presents a snapshot of one of the earliest structures generated in the TTSS assembly process, but also reveals features indicative of its role as the molecular platform for subsequent construction of the secretion apparatus.

Needling Questions

Type III secretion systems are somewhat misleadingly named. This molecular apparatus doesn't simply secrete, leak, or ooze toxins into its environment so much as it mainlines them directly into a host cell. Thus its importance in understanding how bacteria infect healthy cells is obvious, and detailed knowledge of its mechanisms would definitely tip the balance of power toward us humans in our constant battles with the microorganisms that colonize our bodies. More generally, this "nanoneedle" may be of interest as a possible tool for introducing engineered proteins into almost any type of cultured cell. Such applications would require answers to basic questions such as what triggers injection and how proteins are unfolded before passing through the needle. From an evolutionary standpoint, type III secretion systems provide a fascinating case study. They are found in a broad range of bacteria that are both harmful and beneficial and that affect both plant and animal kingdoms. Genetic sequencing studies seem to indicate that type III secretion systems come from a common ancestor foreign to the bacteria. Crystallographic studies such as the one by Yip et al. contribute key pieces of the puzzle, providing new insights into the assembly of the structure's basic building blocks during the earliest stages of the needle's formation.

The TTSS needle complex is found in gram-negative bacteria (e.g. Yersinia, Shigella, Salmonella, Pseudomonas, and E. coli), which are all characterized by a double-membrane cell wall. The needle complex spans the two membranes, with the rigid needle protruding outside the cell. The complex has a base of two rings: a larger one anchored to the inner membrane and a smaller one embedded in the outer membrane. While recent electron microscopy images have revealed the gross morphology of the TTSS, a more detailed analysis of the structural characteristics and organization of these protein components within the bacterial membranes is necessary to understand how protein transfer is mediated and regulated.

To improve the current view of the molecular architecture of the needle complex, the researchers completed the first crystallographic analysis of a basic structural component of the TTSS, that of the protein EscJ from pathogenic E. coli, solved to a resolution of 1.8 Å at ALS Beamline 8.2.1. EscJ belongs to the YscJ/PrgK protein family, members of which are commonly found across many different species of bacteria possessing TTSSs. Most importantly, the self-association ("multimerization") of proteins in this family has been shown to be one of the earliest events in the assembly of the TTSS. The crystal structure of EscJ reveals, in atomic detail, how this interaction can occur.

The unique manner in which EscJ molecules pack when in crystalline form strongly suggests the formation of higher-ordered multimers. In addition, the YscJ/PrgK proteins share similarities in their amino-acid sequences with proteins known, based on earlier electron-microscopy studies, to form a ring. Using the interaction and symmetry information within the crystal, the researchers were able to construct a ring model containing 24 EscJ molecules and demonstrating several features—including distinctive surface grooves and charged patches—indicative of a role as a docking platform. The researchers believe that the bacterium likely produces such a structure as a platform for assembly of the rest of the needle complex. Several pieces of data have provided validation for this model. The dimensions of the modeled ring (~180 Å in diameter and ~52 Å thick) match those previously estimated by electron-microscopy studies. In addition, the number of subunits (24) agrees with results from an extensive labeling and mass-spectrometry analysis performed on isolated needle complexes.

Top left: Ribbon representation of the EscJ monomer, showing two domains. Top right: Arc-shaped EscJ tetramer. Bottom left: Interface between domain 1 of two EscJ monomers, colored green and blue. The residues involved in hydrogen-bonding interactions are highlighted with dotted lines. Bottom right: Interface between domain 2 of two EscJ monomers. Residues from two α helices of the green monomer interact with residues from the β sheet of the blue monomer.

Ribbon and surface representation of the modeled 24-subunit EscJ ring.

This structural analysis of EscJ provides an exciting starting point in the quest to further understand the structure and function of the TTSS. Knowledge of this protein-transfer mechanism will eventually enable the design of novel inhibitors that can combat a broad range of diseases in which the TTSS plays a central role.

Research funding: Natural Sciences and Engineering Research Council of Canada, Michael Smith Foundation for Health Research, Howard Hughes Medical Institute International Scholar Program, Canadian Institutes of Health Research, Canadian Bacterial Diseases Network, and National Institutes of Health. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.